Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The review manuscript entitled “Mitochondrial Regulation of the NLRP3 Inflammasome in Diabetic Kidney Disease: From Mechanisms to Therapeutic Strategies” by Chen X et al. provides a comprehensive overview of the role of the NLRP3 inflammasome in mitochondrial function and highlights the therapeutic potential of targeting the mitochondria–NLRP3 inflammasome axis. The manuscript is generally well written, and the discussion is aligned with current knowledge in the field of diabetic kidney disease (DKD).

I have the following comments and suggestions for the authors:

1. Figure 1: Please clarify whether this figure is original or adapted from a previously published source. If adapted, kindly provide the appropriate citation; if original, please indicate the software or platform used to create it. Additionally, specify the organelle where “post-translational licensing” occurs and consider increasing the font size of the protein labels for better readability.
2. Referencing style: In several instances, multiple references (e.g., 18–25) are grouped at the end of long paragraphs. It would improve clarity to cite references individually within sentences, allowing readers to clearly identify the source of each statement.
3. Abbreviations: Please include a table of abbreviations or ensure that all abbreviations (including those for cell types) are defined upon first use in the text.
4. Figure 2: Please clarify the significance of the two mitochondrial color schemes (pink and blue). Do these represent healthy versus pathological conditions? If so, this should be clearly labeled within the figure.
5. Figure 3: Similar to Figure 1, please indicate whether this figure is original or adapted. Provide appropriate citations if applicable, or specify the drawing platform if it is original. Increasing the size of protein labels would also improve clarity.
6. Rationale: The manuscript would benefit from a clearer explanation of why the NLRP3 inflammasome was specifically selected in the context of DKD, and the importance of focusing on the mitochondria–NLRP3 inflammasome axis. I recommend adding a few sentences to explicitly state the rationale and criteria guiding this focus.

Author Response

Comments 1: [Figure 1: Please clarify whether this figure is original or adapted from a previously published source. If adapted, kindly provide the appropriate citation; if original, please indicate the software or platform used to create it. Additionally, specify the organelle where “post-translational licensing” occurs and consider increasing the font size of the protein labels for better readability.]

Response 1: Thank you for pointing this out. We agree that the subcellular localization of NLRP3 has become an important topic in recent studies, and that the organelle context of NLRP3-related post-translational modifications should be clearly indicated. Accordingly, we carefully revisited the relevant literature and revised Figure 1 to clarify the organelle-associated localization involved in “post-translational licensing” of NLRP3. These revisions can be found on page 47 of the revised manuscript and on the following page of this response letter.

 

Comments 2: [Referencing style: In several instances, multiple references (e.g., 18–25) are grouped at the end of long paragraphs. It would improve clarity to cite references individually within sentences, allowing readers to clearly identify the source of each statement.]

Response 2: Thank you for your valuable suggestion. We have revised the manuscript accordingly. The grouped references at the end of long paragraphs have been redistributed and placed within the relevant sentences so that each citation now more clearly corresponds to the specific statement it supports. This change improves the readability and clarity of the referencing style. Below, we provide the example you mentioned, namely the section involving references 18–25. However, this paragraph has undergone substantial logical restructuring compared with the previous submission; therefore, the excerpt is provided for your reference only.

Comments 3: [Abbreviations: Please include a table of abbreviations or ensure that all abbreviations (including those for cell types) are defined upon first use in the text.]

Response 3: Thank you for your suggestion. We have added a table of abbreviations and ensured that all abbreviations, including cell-type abbreviations, are defined upon first use in the revised manuscript. The abbreviations list is on pages 17 to 21 of the revised version.

Comments 4: [Figure 2: Please clarify the significance of the two mitochondrial color schemes (pink and blue). Do these represent healthy versus pathological conditions? If so, this should be clearly labeled within the figure.]

Response 4: Thank you for this helpful comment. We apologize for the lack of clarity regarding the color scheme used for mitochondria in the figure. The pink mitochondria indicate factors or conditions that promote NLRP3, whereas the blue mitochondria indicate factors or conditions that inhibit NLRP3. To avoid confusion, we have revised the figure by adding clear labels to indicate “Promoting NLRP3” and “Inhibiting NLRP3”. We have also clarified this color coding in the figure legend. These revisions can be found on page 48 of the revised manuscript and on the following page of this response letter.

Comments 5: [Figure 3: Similar to Figure 1, please indicate whether this figure is original or adapted. Provide appropriate citations if applicable, or specify the drawing platform if it is original. Increasing the size of protein labels would also improve clarity.]

Response 5: Thank you for your valuable suggestion. We have increased the font size of the protein labels in the figure to improve readability and clarify in the figure legend that Figure 3 is an original schematic created by the authors using Figdraw.

Comments 6: [Rationale: The manuscript would benefit from a clearer explanation of why the NLRP3 inflammasome was specifically selected in the context of DKD, and the importance of focusing on the mitochondria–NLRP3 inflammasome axis. I recommend adding a few sentences to explicitly state the rationale and criteria guiding this focus.]

Response 6: Thank you for your valuable suggestion. We agree that the rationale for focusing on the NLRP3 inflammasome, particularly the mitochondria–NLRP3 inflammasome axis, should be stated more explicitly. Specifically, we now emphasize that NLRP3 can integrate multiple danger signals, including metabolic stress and oxidative damage, thereby initiating and amplifying inflammatory responses. In addition to its inflammation-related effects, we further highlight that NLRP3 is closely associated with several key pathological processes in DKD, including oxidative stress, lipid dysregulation, and impaired autophagy. We have also added several sentences to explain the importance of the mitochondria–NLRP3 inflammasome axis. We emphasize that mitochondria are among the early affected organelles in DKD and can convert danger signals such as metabolic stress into downstream mechanisms, including mitochondrial reactive oxygen species production and mitochondrial DNA release. These mitochondrial signals can promote NLRP3 activation, thereby contributing to the initiation and progression of DKD. The relevant revisions have been added to the revised manuscript, mainly in the Introduction section and at the beginning of the fourth paragraph.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is a timely and comprehensive review discussing the interplay between mitochondrial dysfunction and NLRP3 inflammasome activation in diabetic kidney disease. The manuscript covers important mechanisms, including mtROS, mtDNA release, mitochondrial dynamics, mitophagy, metabolic reprogramming, pyroptosis, fibrosis, and emerging therapeutic strategies. Overall, the topic is highly relevant to the field, and the manuscript provides a useful synthesis of recent literature.

However, several points should be addressed to improve clarity, organization, and scientific precision.

1. The authors should reduce redundancy across sections. Several concepts, including mtROS, TXNIP/NLRP3 signaling, mitophagy, pyroptosis, and fibrosis, are discussed multiple times in overlapping ways. Streamlining these sections would improve readability.

2. Section 3.4.3 appears to have an incorrect title. Both Sections 3.4.2 and 3.4.3 are titled “Crosstalk Between the NLRP3 Inflammasome and Lipid Dysregulation.” Section 3.4.3 seems to discuss oxidative stress and should be retitled accordingly.

3. The authors should more clearly distinguish DKD-specific evidence from mechanisms inferred from other disease models or non-renal systems. This is especially important for mitochondrial localization of NLRP3, cardiolipin/MAVS/MFN2-mediated recruitment, GSDMD-mediated mitochondrial injury, and some metabolic intermediate-related mechanisms. These mechanisms are interesting, but their relevance to DKD should be presented more cautiously when direct evidence is limited.

4. The figures are informative but quite dense. Figures 2 and 3 include many molecular pathways, which may be difficult for readers to follow. The authors may consider simplifying the figures, improving label readability, or separating core DKD-supported mechanisms from more speculative mechanisms.

5. The therapeutic section is useful, but it would benefit from clearer classification of interventions according to the strength of evidence. For example, direct NLRP3 inhibitors, downstream cytokine/caspase-1 targeting, mitochondrial antioxidants, mitophagy modulators, and natural compounds could be more clearly separated into preclinical, early clinical, and clinically approved categories.

6. The conclusion is very brief compared with the depth of the manuscript. The authors should expand the Summary and Perspectives section to better highlight key knowledge gaps, including cell type-specific mechanisms, human DKD validation, biomarker development, and the need for renal outcome-based clinical trials.

7. The English is generally understandable, but the manuscript would benefit from language polishing. Some sentences are overly long, and there are minor formatting issues, including missing spaces and awkward phrasing.

Author Response

Comments 1: The authors should reduce redundancy across sections. Several concepts, including mtROS, TXNIP/NLRP3 signaling, mitophagy, pyroptosis, and fibrosis, are discussed multiple times in overlapping ways. Streamlining these sections would improve readability.

Response 1: We thank the reviewer for this helpful comment. We agree that several key concepts, including mitochondrial ROS, TXNIP/NLRP3 signaling, mitophagy, pyroptosis, and fibrosis, were discussed in partially overlapping ways in the previous version. To improve the readability and coherence of the manuscript, we have carefully streamlined the relevant sections and reduced redundant descriptions throughout the text. 
Specifically, in Section 3.4.3, “Crosstalk Between the NLRP3 Inflammasome and Oxidative Stress,” we removed the repeated discussion regarding oxidative stress-mediated activation of the NLRP3 inflammasome, as this mechanism is discussed in greater detail in a subsequent section. （page 7）
We also compressed the discussion of mitophagy to avoid reiterating similar mechanisms and, instead, incorporated new content on mitochondrial biogenesis to provide a more balanced overview of mitochondrial quality control. （page 11）
In addition, we reorganized and refined the sections on pyroptosis and fibrosis to improve their internal logic and to minimize repeated explanations of overlapping inflammatory and fibrotic pathways. These revisions have made the manuscript more concise and better structured, while preserving the key mechanistic connections among mitochondrial dysfunction, inflammasome activation, cell death, and fibrotic remodeling. （page 4，5）

 

 

Comments 2: Section 3.4.3 appears to have an incorrect title. Both Sections 3.4.2 and 3.4.3 are titled “Crosstalk Between the NLRP3 Inflammasome and Lipid Dysregulation.” Section 3.4.3 seems to discuss oxidative stress and should be retitled accordingly.

Response 2: Thank you for pointing this out. We apologize for this oversight. We have now revised the title of Section 3.4.3. The updated title is “Crosstalk Between the NLRP3 Inflammasome and Oxidative Stress.”These revisions can be found on page 7 of the revised manuscript.

 

 

Comments 3: The authors should more clearly distinguish DKD-specific evidence from mechanisms inferred from other disease models or non-renal systems. This is especially important for mitochondrial localization of NLRP3, cardiolipin/MAVS/MFN2-mediated recruitment, GSDMD-mediated mitochondrial injury, and some metabolic intermediate-related mechanisms. These mechanisms are interesting, but their relevance to DKD should be presented more cautiously when direct evidence is limited.

Response 3: We thank the reviewer for this insightful and important comment. We fully agree that mechanisms directly supported by DKD-specific evidence should be clearly distinguished from those inferred from other disease models or non-renal systems, especially when discussing mitochondrial localization of NLRP3, cardiolipin/MAVS/MFN2-mediated recruitment, GSDMD-mediated mitochondrial injury, and metabolic intermediate-related mechanisms. In response, we have carefully revised the relevant sections to make this distinction clearer and to present these mechanisms more cautiously when direct evidence in DKD is limited.

We revised the structure and wording of the related sections to explicitly reflect the distinction between DKD-specific findings and evidence derived from non-DKD models. This is also reflected in the revised section titles, such as “4.2 Potential Roles of Mitochondria in NLRP3 Localization and Assembly in DKD” and “4.5 Potential Role of the NLRP3 Inflammasome in Amplifying Mitochondrial Damage.” The use of “potential roles” was intended to avoid overstating mechanisms that remain insufficiently validated in the DKD context.

In the section discussing mitochondria as a potential platform for NLRP3 inflammasome assembly, we reorganized the writing logic and clarified that current DKD studies largely remain at the level of colocalization observations or changes in inflammasome activity after intervention. We now point out that the temporal dynamics, functional necessity, and molecular anchoring mechanisms of NLRP3 mitochondrial recruitment in DKD have not been systematically validated. Therefore, evidence from classical inflammasome models is presented as mechanistic support for interpretation rather than as direct proof in DKD. （page 9，10）

For the metabolic intermediate-related mechanisms, we revised the text by discussing each metabolite separately. For each metabolic intermediate, we first summarize the available DKD-related evidence linking it to renal injury, inflammation, mitochondrial dysfunction, or other pathological changes. We then describe evidence from innate immune cell models regarding its association with NLRP3 inflammasome activation. Based on these two lines of evidence, we cautiously discuss the possible significance of these metabolites as candidate metabolic nodes connecting mitochondrial metabolic disturbance with inflammasome activation. This revised organization is intended to distinguish DKD-specific observations from mechanistic evidence derived from non-DKD inflammatory models, and to avoid implying that all of these mechanisms have been directly demonstrated in DKD. （page 12）

Similarly, in Section 4.5, “Potential Role of the NLRP3 Inflammasome in Amplifying Mitochondrial Damage,” we now explicitly state that direct evidence in the DKD context remains limited, and that the current understanding of GSDMD-mediated mitochondrial injury and related feed-forward mechanisms is largely derived from mechanistic studies in non-DKD models. （page 13）

Overall, these revisions clarify the evidentiary basis of the discussed mechanisms and ensure that DKD-specific conclusions are separated from hypotheses or mechanistic inferences based on other experimental systems. We believe that this has made the manuscript more balanced, rigorous, and appropriately cautious.

 

 

Comments 4: The figures are informative but quite dense. Figures 2 and 3 include many molecular pathways, which may be difficult for readers to follow. The authors may consider simplifying the figures, improving label readability, or separating core DKD-supported mechanisms from more speculative mechanisms.

Response 4: We thank the reviewer for this helpful suggestion. We agree that Figures 2 and 3 contained multiple molecular pathways and may have appeared relatively dense in the previous version, which could make them difficult for readers to follow. Accordingly, we have revised the figures to improve clarity and readability.

Specifically, in Figure 2, we removed several mechanisms that were less directly related to the main focus of the manuscript. In Figure 3, we also made minor content adjustments to simplify the presentation and improve the overall organization of the figure. Importantly, we added specific icons in Figure 3 to distinguish mechanisms supported by DKD-related evidence from those mainly inferred from non-DKD models or other inflammatory systems. This modification is intended to help readers more clearly recognize the evidentiary basis of each mechanism and avoid overinterpreting speculative pathways as DKD-established mechanisms. （page 48，50）

We believe that these revisions make the figures clearer, more accessible, and better aligned with the revised text.

Comments 5: The therapeutic section is useful, but it would benefit from clearer classification of interventions according to the strength of evidence. For example, direct NLRP3 inhibitors, downstream cytokine/caspase-1 targeting, mitochondrial antioxidants, mitophagy modulators, and natural compounds could be more clearly separated into preclinical, early clinical, and clinically approved categories.

Response 5: We thank the reviewer for this helpful suggestion. We agree that the strength of evidence is an important consideration when discussing potential therapeutic strategies. However, because many of the interventions mentioned in this section have not been specifically validated or approved for DKD, we felt that a strict division into preclinical, early clinical, and clinically approved categories might be difficult to apply consistently and could potentially be misleading.

Therefore, we retained the current mechanism-based structure, which separates the interventions according to their main biological targets and modes of action. We believe this organization better matches the overall mechanistic focus of the review, while still allowing readers to appreciate the therapeutic relevance and current limitations of these approaches.

 

 

Comments 6: The conclusion is very brief compared with the depth of the manuscript. The authors should expand the Summary and Perspectives section to better highlight key knowledge gaps, including cell type-specific mechanisms, human DKD validation, biomarker development, and the need for renal outcome-based clinical trials.

Response 6: We thank the reviewer for this valuable suggestion. We agree that the previous “Summary and Perspectives” section was relatively brief compared with the depth of the manuscript. Accordingly, we have expanded this section to provide a more comprehensive discussion of the major knowledge gaps and future directions. In the revised version, we now better highlight several important issues, including cell type-specific mechanisms, the need for validation in human DKD samples, biomarker development, and the importance of renal outcome-based clinical trials. These additions help clarify the remaining challenges in this field and strengthen the translational perspective of the manuscript.

 

Comments 7: The English is generally understandable, but the manuscript would benefit from language polishing. Some sentences are overly long, and there are minor formatting issues, including missing spaces and awkward phrasing.

Response 7: Thank you for your valuable suggestion. We have refined sentence structures, improved word choice, and revised unclear expressions throughout the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

Authors provide a balanced and comprehensive review of mitochondrial regulation of the NLRP3 inflammasome in diabetic kidney disease and discuss therapeutic strategies.

Minor corrections/suggestions

1) Please include MCP-1 in your Figure 1 where IL-1 could promote MCP-1 production.

2) Please note that NOX4 is not a major ROS source while NOX1 and NOX2 may play a more significant pathogenic role. PMID: 24053613

3) mtROS sources and alterations of mitochondrial metabolic and antioxidant pathways are not discussed. It may benefit to mention acetylation pathways. See PMID: 35086984 and PMID: 31852393.

4) It may benefit to cite a previously reported link between Sirt3 deficiency and NLRP3 activation. PMID: 31852393

Author Response

Comments 1: Please include MCP-1 in your Figure 1 where IL-1 could promote MCP-1 production.

Response 1: We highly appreciate the reviewer for pointing out this critical downstream mechanism. We entirely agree that the induction of MCP-1 by secreted IL-1β is a pivotal step that bridges intracellular inflammasome activation to tissue-level immune cell recruitment.
Following your constructive suggestion, we have updated our classical activation model (Figure 1). To maintain the visual focus on the NLRP3 inflammasome machinery while accurately reflecting your point, we have added a downstream signaling branch originating from secreted IL-1β. We have explicitly included MCP-1 (alongside other representative secondary cytokines like IL-6) as the key downstream chemokine responsible for amplifying the inflammatory cascade. These revisions can be found on the revised manuscript. We believe this addition significantly enhances the biological completeness of our model, and we thank the reviewer again for this valuable input.

 

Comments 2: Please note that NOX4 is not a major ROS source while NOX1 and NOX2 may play a more significant pathogenic role. PMID: 24053613

Response 2: Thank you very much for your insightful comment and for bringing this important point to our attention. We fully agree with your view that NOX4 is not generally considered a major source of ROS, whereas NOX1 and NOX2 may play more significant pathogenic roles. We sincerely appreciate your expertise and guidance on this issue.

In the original manuscript, our citation of “NOX4” was mainly based on a previous study showing that NLRP3 knockout could effectively block high glucose-induced Nox4 expression and ROS burst. Therefore, we used this evidence to support the notion that the NLRP3 inflammasome may participate in oxidative stress regulation. However, inspired by your subsequent comment, we agree that the NOX1/2-related content is more appropriate and informative when discussing the sources of mtROS in the pathological context. Accordingly, we have revised the relevant section of the manuscript. Thank you again for your valuable suggestion, which has helped us improve the accuracy and rigor of our manuscript. These revisions can be found at the end of our response to Comment 3 and in the corresponding revised section of the manuscript（page 8）.

 

Comments 3: mtROS sources and alterations of mitochondrial metabolic and antioxidant pathways are not discussed. It may benefit to mention acetylation pathways. See PMID: 35086984 and PMID: 31852393.

Response 3: Thank you very much for pointing out this structural weakness in the manuscript. We agree that the sources of mtROS, as well as alterations in mitochondrial metabolic and antioxidant pathways, were not sufficiently and systematically discussed in the previous version. In revising this section, we considered adding a separate and more detailed discussion of mtROS generation and the associated mitochondrial metabolic pathways. However, a full expansion of these mechanisms would substantially increase the length of the manuscript and might shift the focus toward detailed mitochondrial metabolism rather than the central theme of the review. Therefore, we have adopted a more concise approach: in the first paragraph of the revised section, we integrated the key mechanisms related to mtROS generation and mitochondrial dysfunction without developing them as an independent subsection. In the third paragraph, where potential interventions are discussed, we further highlighted relevant mechanistic pathways, including the Nrf2/antioxidant system and SIRT3-mediated acetylation-related regulation. We sincerely appreciate the reviewer’s insightful suggestion, which helped us improve the balance and structure of this part of the manuscript. We would also be very happy to further revise this section if the reviewer feels that a more detailed or differently organized discussion would be more appropriate.

These revisions can be found in the manuscript（page 8）.

 

Comments 4: It may benefit to cite a previously reported link between Sirt3 deficiency and NLRP3 activation. PMID: 31852393

Response 4: Thank you for your valuable suggestion. We agree that citing the previously reported association between SIRT3 deficiency and NLRP3 inflammasome activation would strengthen the mechanistic support for this section. These revisions can be found at our response to Comment 3.

Author Response File: Author Response.pdf